| Objective: Leukemia is a malignant clonal hematopoietic stem cell disease, the incidence of leukemia is about 2.76/10 million annually .The mortality of Leukemia ranks the sixth among all malignant disease. In children and adult yonger than 35 years old, the leukemia related mortality ranks the top one. In recent years, great progress in the treatment of leukemia has been made not only in traditional chemotherapy, hematopoietic stem cell transplantation, but also in targeted therapy, for example the differentiation induction therapy of acute promyelocytic leukemia with all-trans retinoic acid (ATRA) and the application of tyrosin kinase inhibitor (Glevic) in the treatment of BCR/ABL positive acute lymphoblastic leukemia. However, drug resistance and the inhibition of bone marrow hyperplasia with chemotherapy drugs are still very tough clinical problems, affecting the efficacy of leukemia treatment and prognosis of leukemia patients.Amifostine, also known as Ethiofos, WR-2721 or Ethyol, is the first broad-spectrum internationally recognized cytoprotective agent. It was developed by the U.S. Army Institute of Research firstly, and used for preventing the damage from nuclear radiation and chemical weapons. U.S. FDA approved that Amifostine in 1996 can be used for clinical treatment. It could prevent adverse effects of chemotherapy and protect the bone marrow, kidney, heart and other organs. In terms of the protective effect of Amifostine, alkaline phosphatases way was considered playing a classic pharmacological mechanism. However, in recent years, researchers found that Amifostine has many biological effects, such as functioning as a hematopoietic cell growth factor, inducing tumor cell apoptosis, inhibiting tumor cell cycle progressing anti-angiogenesis and anti-tumor effect. The mechanism of these effects can not be explained by its activity of alkaline phosphatase. And whether Amifostine has the same effect on leukemia cells and bone marrow stromal cells is not clear. Therefore, in the present study, Dami (human acute mega- karyocytic leukemia cell line cells), HL-60(acute promyelocytic leukemia cell line cells) and BMSC (bone marrow stromal cell) were treat with Amifostine at different concentrations and for different time period to observe the effect of Amifostine on cell proliferation, cell cycle distribution, Apoptosis, and to evaluate the mechanism of these biological effects of Amifostine. These cells were also treated with Amifostine and cytarabine (Ara-C) or vincristine (VCR) to see whether there is any synergestic effect between Amifostine and Ara-C or VCR and whether there is preventing effect of Amifostine for BMSC damage induced by Ara-C or VCR.Methods:Proliferation inhibition effect of Amifostine on Dami, HL-60 and BMSC was measured by CCK-8; Cell morphological changes induced by Amifostine was observed under a inverted microscope; The percentage of apoptotic cells was examined by flow cytometry of the Amifostine treated and Annexin V-FITC/PI double stained Dami, HL-60 and BMSC ; Cell cycle distribution and DNA ploidy changes was evaluated with PI staining and flow cytometry; The mRNA expression level of apoptosis related gene bcl-2, caspase -3, cell cycle related genes cyclinA, cyclinB1, cyclinD1 and megakaryocytic differentiation associated gene GATA-1, NF-E2 mRNA.Results:1 After treated with Amifostine for 24h, 48h, 72h, at the final concentrations of 0.35mg/ml, 0.75mg/ml, and 1.5mg/ml, the Dami and HL-60 cell viability significantly decreased compared with that in control group, especially at the concentration of 1.5mg/ml group, inhibition ratios were 56.42%, 89.72% anad 92.08%, respectively, in Dami and 43.86%, 80.47%, 86.5%, respectively, in HL60 when the cells were treated with Amifostine for 48h. The IC50 was 0.25mg/ml and 0.37mg/ml Amifostine for Dami and HL-60, espectively, when the cells were treated for 48h. However, Amifostine at the same concentration range and the same treatment time had no effect on the proliferation of BMSC.2 After the treatment with Amifostine at the concentration of 0.35mg/ml, 0.75mg/ml and1.5mg/ml for 24h and 48h, the apoptosis rate of Dami was 9.123±3.766%,11.320±1.530%,11.890±3.200%,17.543±3.365% and 6.727±3.126%,20.203±5.115%,30.023±6.433%,63.397±24.064%. The apoptosis rate of Dami was increased in a dose and time dependent.HL-60 was same. While, treatement with Amifostine at the same concentration for the same 24h and 48h, no apoptosis sign was observed in BMSC.3 After the treatment with Amifostine at the concentration of 0.35 mg / ml for 24h,48h, the percent of Dami cells in G0/G1 phase was from 46.28%,43.57% to 14.19% and 4.27% and the percent of G2 / M phase cells was from 13.39%,15.65% to 68.20% and 67.82%.4 Amifostine at low concentration of (0.35mg/ml) had no synergetic effect with Ara-C in cell proliferation inhibition, while enhanced the growth suppressive effect of vincristine on the two cell lines. Amifostine could reduce the inhibitory effect of vincristine on BMSC, but failed to prevent BMSC suppression induced with Ara-C treatment.5 Amifostine at low concentration of (0.35mg/ml) had no synergetic effect with cytarabine or vincristine in inducing Dami, HL-60 cells apoptosis.6 After Dami had been treated with Amifostine (0.75mg/ml and1.5mg/ml) for 24h, the expression of bcl-2 decreased 0.307 and 0.532 times compared with that of control group (P<0.05). After Dami had been treated with Amifostine(1.5mg/ml) for 24h, the expression of caspase-3 increased 5.047 times compared with that of control group (P<0.05). the expression of cyclinD1 at the concentration of 0.35mg/ml, 0.75mg/ml increased 2.78和1.856 times compared with that of control group (P<0.05). the expression of cyclinB1,GATA-1, NF-E2 mRNA had no change compared with that of control group.Conclusions : Amifostine could inhibit Dami and HL-60 cells proliferation, promote their apoptosis and enhance the anti-leukemia effect of vincristine. The mechanism of such effect might be related with its arresting cell cycle at G2/M phase, downregulating the expression of anti-apoptosis gene bcl-2 and the cycle related genes cyclinA, cyclinB1, cyclinD1, but upregulating the expression of pro-apoptosis gene caspase-3. Amifostine had no effect on BMSC proliferation and apoptosis, but can reduce the inhibitory effect of vincristine on BMSC.
|
PDF Full Text Request